CN107190004A - A kind of mevalonate pathway of TIGR regulation and control - Google Patents

A kind of mevalonate pathway of TIGR regulation and control Download PDF

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CN107190004A
CN107190004A CN201610142672.5A CN201610142672A CN107190004A CN 107190004 A CN107190004 A CN 107190004A CN 201610142672 A CN201610142672 A CN 201610142672A CN 107190004 A CN107190004 A CN 107190004A
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mev
luteole
seq
tigr
sequences
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辛珉
刘建忠
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Abstract

The invention belongs to biochemical field, it is related to one kind using TIGR (tetracycline-regulated gene region) regulation and control MEV (mevalonic acid) approach, produces isoamyl vinyl compound, especially luteole.Specifically, in the engineering colon bacillus (ZEAX) that external source MEV approach is incorporated into production luteole, especially into the TIGR MEV regulated and controled upstream pathway and downstream pathway, to coordinate the generation of intermediate product, reduce intermediate accumulation and toxicity, the final yield for improving luteole are produced to cell.

Description

A kind of mevalonate pathway of TIGR regulation and control
Technical field
The invention belongs to biochemical field, and in particular to utilize Escherichia coli recombination engineering, produce luteole.
Background technology
Luteole is a kind of carotenoid of high value, except as health products, cosmetics, food etc., clinically also wide It is general to be used to treat treating senile maculopathy.Constantly expand with the market demand of luteole, the mode of production of former luteole (chemical synthesis is purified with direct from plant) is difficult to stable supply market.Therefore, it is badly in need of improving the yield of luteole.
Existing multiple seminars report at present can produce luteole (Albrecht et with engineering colon bacillus al.Biotechnol Lett 1999,21(9):791–795；Nishizaki et al.,Appl Environ Microbiol 2007；73(4):1355–1361；Li et al.,J Ind Microbiol Biot.2015,42,627-636).Wherein Li The luteole yield highest of the engineering colon bacillus of et al. reports, reaches 11.95mg/g DCW (bacterium dry weight).
Luteole is generated using Escherichia coli, is typically elder generation synthetic mesophase material IPP (Isoprenoid), regeneration Into luteole.
IPP (Isoprenoid) for not be only luteole precursor, or many terpenoids precursor, such as The monoterpenes such as the carotenoid such as lycopene, carrotene, luteole, astaxanthin, limonene and firpene, hair alkene, The type sesquiterpene class compound such as heerabolene and AD, the diterpenoids compound such as Japanese yew alkene etc..
Escherichia coli pass through DXP approach (Fig. 1) by glycolytic pathway intermediate product glyceraldehyde 3-phosphate and pyruvic acid condensation To synthesize IPP.And some eukaryotics such as yeast, then it is that IPP is synthesized by mevalonic acid (MEV) approach (Fig. 1).
Those skilled in the art's research is found, the MEV approach of saccharomyces cerevisiae is introduced in Escherichia coli, by increasing capacitance it is possible to increase IPP confession Give, so as to improve the production efficiency of target product isoamyl vinyl compound.
However, their introduced MEV approach do not consider each intergenic coordinated expression problem, MEV in approach Expressed in approach between gene it is uncoordinated cause MEV approach intermediate accumulations, to cell produce toxicity.So far, do not have yet MEV approach is introduced into Escherichia coli, for producing luteole.
TIGR (tunable intergenic regions, regulatable gene region) is one section between gene and gene of non-volume Code sequence, using TIGR, the expression of multiple genes can be cooperateed with simultaneously.Pfleger etc. reports a kind of TIGR (between regulatable gene Septal area) metabolic pathway technology, i.e., insertion TIGR can coordinate the expression of gene well between two genes, and they utilize the technology Being inserted the gene of saccharomyces cerevisiae MEV upstream pathways after TIGR, its amount for synthesizing mevalonic acid is set to carry 7 times of (Nature of original text Biotechnology 2006,24:1027-1032)。
The content of the invention
Present invention aims at a kind of recombination fragment of TIGR regulation and control MEV approach is provided, the recombination piece is utilized Section can improve IPP yield.
The present invention also aims to provide a kind of production luteole Escherichia coli containing above-mentioned recombinant fragment, institute is utilized The Escherichia coli stated can improve the yield of luteole.
The purpose of the present invention is realized by following technological means：
The invention provides a kind of TIGR sequences.
The sequence is selected from the TIGR3 sequences as described in SEQ ID No.3 or the TIGR4 sequences as described in SEQ ID No.4 Row.
The invention provides the recombination fragment that a kind of TIGR regulates and controls MEV approach.
Described MEV approach includes MEV upstream pathways gene and MEV downstream pathway genes.
Described MEV upstream pathway genes include：
atoB：Acetyl coenzyme A acetyl transferase gene；
HMGS：HMG CoA synthase gene；
HMGR：3-hydroxy-3-methylglutaryl coenzyme A reductase gene；
TIGR1 sequences are inserted between described MEV upstream pathway genes atoB and HMGS, are inserted between HMGS and HMGR TIGR2 sequences.
Described TIGR1 is as shown in SEQ ID No.1；
Described TIGR2 is as shown in SEQ ID No.2；
Described MEV downstream pathway genes include：
MK：Mevalonate kinase gene；
PMK：Phosphomevalonate kinase gene；
MVD：Pyrophosphoric acid mevalonic acid decarboxylase gene.
TIGR3 sequences are inserted between described MEV downstream pathway gene Ms K and PMK, TIGR4 sequences are inserted between PMK and MVD Row.
Described TIGR3 is as shown in SEQ ID No.3；
Described TIGR4 is as shown in SEQ ID No.4.
The TIGR sequences both can be through fully synthetic, it would however also be possible to employ the library approach screening of the report such as Pfleger is obtained (Nature Biotechnology 2006,24:1027-1032)。
The recombination fragment for the TIGR regulation and control MEV approach that the present invention is provided also includes promoter P1.
Described promoter P1 can be that the Escherichia coli such as P37, PgadE, Ptrc, Ptac, PBAD and PlacUV5 start Son；Preferably, it is PgadE.
The recombination fragment for the TIGR regulation and control MEV approach that the present invention is provided also includes idi and ispA genes.
The TIGR regulation and control MEV approach that the present invention is provided is as shown in Figure 2.
First gene atoB of described MEV approach comes from Escherichia coli；Other genes (atoB, HMGS, HMGR, MK, PMK, MVD, idi, ispA) it may be from saccharomyces cerevisiae, other microorganisms, such as aurococcus can also be come (Staphylococcus aureus), Bordetella (Bordetella petrii), acide eating Darfot bacteria (Delftia ) and mevalonic acid pseudomonad (Pseudomonas mevalonii) etc. acidovorans.Both can be natural gene or can To be its mutator.Both can all from a kind of mentioned microorganism, can be from above two or multiple-microorganism Combination.
MK, PMK and MVD are selected from saccharomycete, respectively ERG12, ERG8 and ERG19 in an embodiment of the present invention.
Regulate and control the recombination fragment of MEV approach using the TIGR of the present invention, available for the isoamyl alkenes that production IPP is precursor The Escherichia coli of compound, including carotenoid, limonene and the firpene such as lycopene, carrotene, luteole, astaxanthin Deng monoterpene, the type sesquiterpene class compound such as hair alkene, heerabolene and AD, the diterpenoids such as Japanese yew alkene Compound etc..In the preferred embodiment of the invention, for producing luteole.
The invention provides it is a kind of containing TIGR regulate and control MEV approach recombination fragment process for preparing plasmid, specifically such as Under：
The structure of the MEV upstream pathways of S1.TIGR regulation and control
1) by primer of F1/F2, Escherichia coli Escherichia coli DH5Genome is template, PCR amplifications AtoB genes, are connected on pBAD33 carriers, obtain pBAD33-atoB；
2) by primer of F3/F4, saccharomyces cerevisiae (Saccharomyces cerevisiae) genome be template, PCR expands Increase HMGS genes, be connected on pMD18-T carriers, obtain pMD-HMGS；
3) by primer of F5/F6, saccharomyces cerevisiae (Saccharomyces cerevisiae) genome be template, PCR expands Increase tHMGR genes, be connected on pMD18-T carriers, obtain pMD-tHMGR；
4) by primer of F7/F8, pMD-HMGS be template, PCR amplifications；By primer of F9/F6, pMD-tHMGR be template, PCR is expanded；
5) by primer of F7/F6,2 PCR fragments of previous step glue reclaim be masterplate, PCR amplifications are connected to pBAD33- Between atoB SmaI/PstI, pBAD33-MevTTIGR is obtained.
6) NheI/PstI digestions pBAD33-MevTTIGR, is connected between pZSBP corresponding restriction enzyme site, obtains pZS- MevTTIGR.
SEQ ID NO.5
F1:GCTGAGCTCTTTCGGAATTAAAGGAGCATCAAATATGAAAAATTGTGTCATCGTCAG(SacI)
SEQ ID NO.6
F2:GATCCCGGGTTAATTCAACCGTTCAATCACC(SmaI)
SEQ ID NO.7
F3:TCAGGATACAGTATCTGCGGTACCGGAGGACAGCTAAATGAAACTGTCCACTAAACTGT
SEQ ID NO.8
F4:GGGTGGTCGCGCACCGGGATCAGGAGATCTTGCTAGGCTTATTTTTTAACATCGTAAGAT
SEQ ID NO.9
F5:TATCGTCGCCTCCGAGACACCATCATTGTATAGGCGGAGGATTACACTATGGACCAACTGGTGAAA ACTG
SEQ ID NO.10
F6:GCTACTGCAGTTAGGATTTAATGCAGGTGACGG(PstI)
SEQ ID NO.11
F7:TCTCCCGGGGCCTAGCAAGATCTCCTGATCAGGATACAGTATCTGCGGTACCG(SmaI)
SEQ ID NO.12
F8:GGCGACGATACGCCAATCCTCAGACTGGCCCAGACTATGCAGATGTCCGGGTGGTCGCGCACCGGG ATCAGG
SEQ ID NO.13
F9:ATCTGCATAGTCTGGGCCAGTCTGAGGATTGGCGTATCGTCGCCTCCGAGAC
S2.MEV downstream pathways pBAD24-MevBIS structure
1) respectively by primer of F10/F11, F12/F13 and F14/F15, saccharomyces cerevisiae (Saccharomyces Cerevisiae) genome is template, and PCR amplification ERG12, ERG19 and ERG8 genes are connected on pBAD24, obtained pBAD24-MevB。
SEQ ID NO.14
F10:CTAGCTAGCTTTCGGAATTAAAGGAGCATCAAATATGTCTCTGCCGTTCCTG(NheI)
SEQ ID NO.15
F11:CTACCCGGGAAACTCGAGTTATGAAGTCCATGGTAAATTCG(SmaI/XhoI)
SEQ ID NO.16
F12:GATCCCGGGTTTCGGAATTAAAGGAGCATCAAATATGACCGTTTACACGGCATCC(SmaI)
SEQ ID NO.17
F13:TGCCTGCAGCCAATCGATTTATTTCTTTGGTAGACCAG(PstI/ClaI)
SEQ ID NO.18
F14:ATTCTCGAGAAAAGGGCCCTTTCGGAATTAAAGGAGCATCAAATATGTCTGAGCTGCGTGCCTTC TCTGCCCCAGGT(XhoI/ApaI)
SEQ ID NO.19
F15:ATTCCCGGGAAAAACTAGTTTATTTATCAAGATAAGTTTCCGGA(SmaI/SpeI)
2) by primer of F16/F17, genome of E.coli be template, PCR amplification idi genes are connected to pMD-18T On, and point mutation removes SpeI restriction enzyme sites, obtains pMD-idi.
SEQ ID NO.20
F16:ATCGATTTTCGGAATTAAGGAGGTAATAAATATGCAAACGGAACACGTCATT(ClaI)
SEQ ID NO.21
F17:GTCGACAAAAGATCTTTATTTAAGCTGGGTAAATGC(PstI/BglII)
3) by primer of F18/F19, genome of E.coli be template, PCR amplification ispA genes are cloned into pMD-idi BglII/PstI between, and simultaneously point mutation remove NheI and SmaI restriction enzyme sites, obtain pMD-idi-ispA.Then ClaI/ is used PstI digestion pMD-idi-ispA, and idi-ispA fragments are connected on pBAD24-MevB, obtain pBAD24-MevBIS.
SEQ ID NO.22
F18:TTAGTCGACTTTCGGAATTAAGGAGGTAATAAATATGGACTTTCCGCAGCAACTCG(BamHI)
SEQ ID NO.23
F19:TTAAGCATGCTTATTTATTACGCTGGATGATG(PstI)
S3.TIGR regulates and controls the structure of MEV downstream pathways
1) following TIGR sequences are inserted in pBAD24-MevBIS, pBAD24-MevBTIGRIS is obtained
TIGR sequences are SEQ ID NO.3 between ERG12 and ERG8 genes：
TIGR3:5'-GCCTAGCAAGATCTCCTGATCCCGGTGCGCGACCACCCGGACATCTGCATAGTCTGGGTG GATCAGGTACACTTACACTTGCCTTGAATTTACAGTATTTCAGTTACCGCTCTATCCTTATCCTTATCCGCTCAAGA TAACCGGATACCGGCCCGATCGGTACCGCAGATACTGAATCC-3'
TIGR sequences are SEQ ID NO.4 between ERG8 and ERG19 genes：
TIGR4:5'-GCCTAGCAAGATCTCCTGATCCACCTTTGATGGCTAGAAAAATTAAGCTGCGGACATCTG CATAGTCTGGGCCAGTCTGAGGACTGGCGGATCAGGGCCTTGAATTTACAGTATTTAATGAACTAGCGTTCCGAGTG CATGCCTTATCCGCTCAAGACATGCACTCGGAACGCATCTAGGGTACCGCAGATACTGTATCC-3'
Above-mentioned TIGR sequences both can be through fully synthetic, it would however also be possible to employ the library approach screening of the report such as Pfleger is obtained (Nature Biotechnology 2006,24:1027-1032)。
2) NheI/PstI digestion pBAD24-MevBTIGRIS are used, is connected between pZSBP corresponding restriction enzyme site, obtains pZS-MevBTIGRIS。
S4.TIGR regulates and controls the structure of MEV approach
1) AvrII/SalI digestion pZS-MevBTIGRIS are used, is connected between pZS-MevTTIGR XbaI/SalI, obtains To TIGR regulation and control MEV approach plasmids pZS-MevTTIGR-MevBTIGRIS.
2) by primer of F20/21, genome of E.coli be template, PCR amplification genes gadE promoter is connected to Between pZS-MevTTIGR BglII/NheI, pZSPgadE-MevTTIGR is obtained.Then by method 1), with pZS- MevBTIGRIS is assembled, and obtains pZSPgadE-MevTTIGR-MevBTIGRIS.
SEQ ID NO.24
F20:CAACAGATCTTTAATACTCTCTCCGCTACG(BglII)
SEQ ID NO.25
F21:ATATACGCTAGCTCGTTTCGAATATGTCATCC(NheI)
In the recombination fragment of the present invention, the output increased lifting of the presence of escherichia coli promoter for luteole Act on.Inventor's research discovery, the presence of escherichia coli promoter, the lifting for having 58%-71% for yield.Described is big Enterobacteria can be the escherichia coli promoters such as P37, PgadE, Ptrc, Ptac, PBAD and PlacUV5.Currently preferred reality Apply in example, promoter preferably is PgadE.
The invention provides a kind of production luteole large intestine for the recombination fragment for regulating and controlling MEV approach containing above-mentioned TIGR Bacterium.
In described production luteole Escherichia coli TIGR regulate and control MEV approach recombination fragment be can with plasmid come Express, progress expression on chromosome can also be incorporated into
Described production luteole Escherichia coli are ZEAX (Li XR et al., J Ind Microbiol Biot.2015,42,627-636).
Described Escherichia coli production luteole (refers to Li XR et al., J Ind using shaker fermentation training method Microbiol Biot.2015,42,627-636).
The beneficial effect that the present invention is obtained:
External source MEV approach is incorporated into the engineering colon bacillus of production luteole by the present invention first, is introduced when especially The MEV approach of TIGR regulation and control.And the whole approach of MEV (3 genes and 3 genes in downstream that include upstream) is adjusted simultaneously Control, the expression of coordinated balance gene, so that the intermediate accumulation of the mevalonate pathway avoided and its poison to Escherichia coli Property.In addition, the MEV approach of TIGR regulation and control significantly improves intermediate product IPP yield, so as to improve using IPP as precursor Luteole yield.
Brief description of the drawings
Fig. 1 Isoprenoid route of synthesis
atoB:Acetyl coenzyme A acetyl transferase gene；HMGS:HMG CoA synthase gene；HMGR:Hydroxyl Methylglutaryl A reductase gene；MK(ERG12):Mevalonate kinase gene；PMK(ERRG8):Phosphomelovanate Kinase gene；MVD(ERG19):Pyrophosphoric acid mevalonic acid decarboxylase gene.
Fig. 2 TIGR regulate and control MEV approach
Embodiment
Technical scheme is further illustrated below by way of specific embodiment, specific embodiment is not represented to this hair The limitation of bright protection domain.Some nonessential modifications and adjustment that other people are made according to theory of the present invention still fall within this The protection domain of invention.
In this programme, the molecular biology experiment technology used includes PCR amplifications, plasmid extraction, plasmid conversion, DNA Fragment connection, digestion, gel electrophoresis etc. all using conventional method, for details, reference can be made to《Molecular Cloning:A Laboratory guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated, 2002, Beijing：Scientific publication Society).
The structure of the MEV upstream pathways of embodiment 1TIGR regulation and control
1) by primer of F1/F2, Escherichia coli Escherichia coli DH5Genome is template, PCR amplifications atoB Gene, is connected on pBAD33 carriers, obtains pBAD33-atoB；
2) by primer of F3/F4, saccharomyces cerevisiae (Saccharomyces cerevisiae) genome be template, PCR expands Increase HMGS genes, be connected on pMD18-T carriers, obtain pMD-HMGS；
3) by primer of F5/F6, saccharomyces cerevisiae (Saccharomyces cerevisiae) genome be template, PCR expands Increase tHMGR genes, be connected on pMD18-T carriers, obtain pMD-tHMGR；
4) by primer of F7/F8, pMD-HMGS be template, PCR amplifications；By primer of F9/F6, pMD-tHMGR be template, PCR is expanded；
5) by primer of F7/F6,2 PCR fragments of previous step glue reclaim be masterplate, PCR amplifications are connected to pBAD33- Between atoB SmaI/PstI, pBAD33-MevTTIGR is obtained.
6) NheI/PstI digestions pBAD33-MevTTIGR, is connected between pZSBP corresponding restriction enzyme site, obtains pZS- MevTTIGR
SEQ ID NO.5
F1:GCTGAGCTCTTTCGGAATTAAAGGAGCATCAAATATGAAAAATTGTGTCATCGTCAG(SacI)
SEQ ID NO.6
F2:GATCCCGGGTTAATTCAACCGTTCAATCACC(SmaI)
SEQ ID NO.7
F3:TCAGGATACAGTATCTGCGGTACCGGAGGACAGCTAAATGAAACTGTCCACTAAACTGT
SEQ ID NO.8
F4:GGGTGGTCGCGCACCGGGATCAGGAGATCTTGCTAGGCTTATTTTTTAACATCGTAAGAT
SEQ ID NO.9
F5:TATCGTCGCCTCCGAGACACCATCATTGTATAGGCGGAGGATTACACTATGGACCAACTGGTGAAA ACTG
SEQ ID NO.10
F6:GCTACTGCAGTTAGGATTTAATGCAGGTGACGG(PstI)
SEQ ID NO.11
F7:TCTCCCGGGGCCTAGCAAGATCTCCTGATCAGGATACAGTATCTGCGGTACCG(SmaI)
SEQ ID NO.12
F8:GGCGACGATACGCCAATCCTCAGACTGGCCCAGACTATGCAGATGTCCGGGTGGTCGCGCACCGGG ATCAGG
SEQ ID NO.13
F9:ATCTGCATAGTCTGGGCCAGTCTGAGGATTGGCGTATCGTCGCCTCCGAGAC
Embodiment 2MEV downstream pathways pBAD24-MevBIS
1) respectively by primer of F10/F11, F12/F13 and F14/F15, saccharomyces cerevisiae (Saccharomyces Cerevisiae) genome is template, and PCR amplification ERG12, ERG19 and ERG8 genes are connected on pBAD24, obtained pBAD24-MevB。
SEQ ID NO.14
F10:CTAGCTAGCTTTCGGAATTAAAGGAGCATCAAATATGTCTCTGCCGTTCCTG(NheI)
SEQ ID NO.15
F11:CTACCCGGGAAACTCGAGTTATGAAGTCCATGGTAAATTCG(SmaI/XhoI)
SEQ ID NO.16
F12:GATCCCGGGTTTCGGAATTAAAGGAGCATCAAATATGACCGTTTACACGGCATCC(SmaI)
SEQ ID NO.17
F13:TGCCTGCAGCCAATCGATTTATTTCTTTGGTAGACCAG(PstI/ClaI)
SEQ ID NO.18
F14:ATTCTCGAGAAAAGGGCCCTTTCGGAATTAAAGGAGCATCAAATATGTCTGAGCTGCGTGCCTTC TCTGCCCCAGGT(XhoI/ApaI)
SEQ ID NO.19
F15:ATTCCCGGGAAAAACTAGTTTATTTATCAAGATAAGTTTCCGGA(SmaI/SpeI)
2) by primer of F16/F17, genome of E.coli be template, PCR amplification idi genes are connected to pMD-18T On, and point mutation removes SpeI restriction enzyme sites, obtains pMD-idi.
SEQ ID NO.20
F16:ATCGATTTTCGGAATTAAGGAGGTAATAAATATGCAAACGGAACACGTCATT(ClaI)
SEQ ID NO.21
F17:GTCGACAAAAGATCTTTATTTAAGCTGGGTAAATGC(PstI/BglII)
3) by primer of F18/F19, genome of E.coli be template, PCR amplification ispA genes are cloned into pMD-idi BglII/PstI between, and simultaneously point mutation remove NheI and SmaI restriction enzyme sites, obtain pMD-idi-ispA.Then ClaI/ is used PstI digestion pMD-idi-ispA, and idi-ispA fragments are connected on pBAD24-MevB, obtain pBAD24-MevBIS.
SEQ ID NO.22
F18:TTAGTCGACTTTCGGAATTAAGGAGGTAATAAATATGGACTTTCCGCAGCAACTCG(BamHI)
SEQ ID NO.23
F19:TTAAGCATGCTTATTTATTACGCTGGATGATG(PstI)
Embodiment 3TIGR regulates and controls the structure of MEV downstream pathways
1) following TIGR sequences are inserted in pBAD24-MevBIS, pBAD24-MevBTIGRIS is obtained
TIGR sequences are SEQ ID NO.3 between ERG12 and ERG8 genes：
TIGR3:5'-GCCTAGCAAGATCTCCTGATCCCGGTGCGCGACCACCCGGACATCTGCATAGTCTGGGTG GATCAGGTACACTTACACTTGCCTTGAATTTACAGTATTTCAGTTACCGCTCTATCCTTATCCTTATCCGCTCAAGA TAACCGGATACCGGCCCGATCGGTACCGCAGATACTGAATCC-3'
TIGR sequences are SEQ ID NO.4 between ERG8 and ERG19 genes：
TIGR4:5'-GCCTAGCAAGATCTCCTGATCCACCTTTGATGGCTAGAAAAATTAAGCTGCGGACATCTG CATAGTCTGGGCCAGTCTGAGGACTGGCGGATCAGGGCCTTGAATTTACAGTATTTAATGAACTAGCGTTCCGAGTG CATGCCTTATCCGCTCAAGACATGCACTCGGAACGCATCTAGGGTACCGCAGATACTGTATCC-3'
Above-mentioned TIGR sequences both can be through fully synthetic, it would however also be possible to employ the library approach screening of the report such as Pfleger is obtained (Nature Biotechnology 2006,24:1027-1032)。
2) NheI/PstI digestion pBAD24-MevBTIGRIS are used, is connected between pZSBP corresponding restriction enzyme site, obtains pZS-MevBTIGRIS。
Embodiment 4TIGR regulates and controls the structure of MEV approach
1) AvrII/SalI digestion pZS-MevBTIGRIS are used, is connected between pZS-MevTTIGR XbaI/SalI, obtains TIGR regulation and control MEV approach plasmids pZS-MevTTIGR-MevBTIGRIS.
2) by primer of F20/21, genome of E.coli be template, PCR amplification genes gadE promoter is connected to Between pZS-MevTTIGR BglII/NheI, pZSPgadE-MevTTIGR is obtained.Then by method 1), with pZS- MevBTIGRIS is assembled, and obtains pZSPgadE-MevTTIGR-MevBTIGRIS.
SEQ ID NO.24
F20:CAACAGATCTTTAATACTCTCTCCGCTACG(BglII)
SEQ ID NO.25
F21:ATATACGCTAGCTCGTTTCGAATATGTCATCC(NheI)
The method that embodiment 5 produces luteole
By TIGR regulation and control MEV approach the plasmids pZS-MevTTIGR-MevBTIGRIS and pZSPgadE- in embodiment 4 MevTTIGR-MevBTIGRIS is transformed into Escherichia coli ZEAX (Li XR et al., the J Ind of production luteole respectively Microbiol Biot.2015,42,627-636), and the method introduced by this article carries out shaking table culture fermentation, analyzes its conjunction Into the situation of luteole, 1 the results are shown in Table.
The difference MEV approach of table 1 synthesizes the influence of luteole to Escherichia coli
As seen from Table 1, the ability that external source mevalonate pathway can promote Escherichia coli to synthesize luteole is introduced, and The TIGR of PgadE drivings regulates and controls the best results of MEV approach, and the yield of its luteole is 22.48mg/g DCW, is Li et Al (Li XR et al., J Ind Microbiol Biot.2015,42,627-636) report twice ((11.95 ± 0.21mg/g DCW).As can be seen here, the solution of the present invention significantly improves the yield of luteole.

Claims (9)

1. a kind of TIGR sequences, selected from the TIGR3 sequences as described in SEQ ID No.3 or the TIGR4 as described in SEQ ID No.4 Sequence.
2. a kind of TIGR regulates and controls the recombination fragment of MEV approach, it is characterised in that regulate and control MEV upstream pathway genes comprising TIGR With MEV downstream pathway genes；
Described MEV upstream pathway genes are included：AtoB, HMGS, HMGR；
TIGR1 sequences are inserted between the atoB and HMGS, TIGR2 sequences are inserted between HMGS and HMGR；
Described TIGR1 is as shown in SEQ ID No.1；
Described TIGR2 is as shown in SEQ ID No.2；
Described MEV downstream pathway genes include：MK, PMK, MVD；
TIGR3 sequences are inserted between described MK and PMK, TIGR4 sequences are inserted between PMK and MVD.
3. recombination fragment as claimed in claim 1, its feature is also being selected from comprising promoter P1, described promoter P1 One kind in escherichia coli promoter P37, PgadE, Ptrc, Ptac, PBAD, PlacUV5；Preferably, it is PgadE.
4. recombination fragment as claimed in claim 1, its feature is also including gene idi and ispA.
5. recombination fragment as claimed in claim 1, it is characterised in that described atoB gene sources are in Escherichia coli.
6. a kind of preparation method of the recombinant plasmid comprising recombination fragment as claimed in claim 1, it is characterised in that bag Containing following steps：
The structure of the MEV upstream pathways of S1TIGR regulation and control；
The structure of S2MEV downstream pathways；
S3TIGR regulates and controls the structure of MEV downstream pathways；
S4TIGR regulates and controls the structure of MEV approach.
7. recombination fragment as claimed in claim 1, it is characterised in that it can be for producing the isoamyl using IPP as precursor Vinyl compound, described isoamyl vinyl compound is lycopene, carrotene, luteole, astaxanthin, limonene, firpene Send out alkene, heerabolene, amorpha-4,11-diene or Japanese yew alkene；Preferably, for producing luteole.
8. a kind of production luteole Escherichia coli containing recombination fragment as claimed in claim 1, it is characterised in that institute The production luteole Escherichia coli stated are ZEAX.
9. Escherichia coli as claimed in claim 8, it is characterised in that its recombination fragment contained can be with plasmid come table Reach, or be incorporated on chromosome and expressed and produce luteole.
CN201610142672.5A 2016-03-14 2016-03-14 A kind of mevalonate pathway of TIGR regulation and control Pending CN107190004A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109666683A (en) * 2019-02-27 2019-04-23 昆明理工大学 Acetyl coenzyme A acetyl transferase gene RKAcaT2 and its application
CN114540363A (en) * 2022-02-17 2022-05-27 江南大学 Construction and protein rapid purification method of human-like collagen recombinant pichia pastoris engineering bacteria
CN114854780A (en) * 2022-04-13 2022-08-05 江南大学 Method for efficiently synthesizing riboflavin based on balanced gene expression

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109666683A (en) * 2019-02-27 2019-04-23 昆明理工大学 Acetyl coenzyme A acetyl transferase gene RKAcaT2 and its application
CN109666683B (en) * 2019-02-27 2021-10-29 昆明理工大学 Acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof
CN114540363A (en) * 2022-02-17 2022-05-27 江南大学 Construction and protein rapid purification method of human-like collagen recombinant pichia pastoris engineering bacteria
CN114540363B (en) * 2022-02-17 2024-05-28 江南大学 Construction and protein rapid purification method of human-like collagen recombinant pichia pastoris engineering bacteria
CN114854780A (en) * 2022-04-13 2022-08-05 江南大学 Method for efficiently synthesizing riboflavin based on balanced gene expression

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